Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells
Dieuwke L. Marvin, Li You, Laura Bornes, Maarten van Dinther, Niek A. Peters, Hao Dang, Sarah K. Hakuno, Marten Hornsveld, Onno Kranenburg, Jacco van Rheenen, Jos H. T. Rohling, Miao‐Ping Chien, Peter ten Dijke, Laila Ritsma
Abstract
Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.